Short peptide affinity tags have become indispensable in protein research. They cannot only be used for affinity purification but also for detection and assay of any fused recombinant protein without the need for any prior knowledge of its biochemical properties. The affinity tag Strep-tag®II (Schmidt & Skerra, Nature Protocols 2 (2007), 1528-1535; U.S. Pat. No. 5,506,121, having the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, SEQ ID NO: 100) is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The currently most efficient streptavidin based receptor for the Strep-tag®II affinity tag are streptavidin muteins with improved binding affinity that are named Strep-Tactin® (Voss & Skerra, Protein Engineering 10 (1997), 975-982; U.S. Pat. No. 6,103,493 or European Patent 0 835 934). The Strep-tag®II binds to the biotin binding pocket enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the affinity resins. The Strep-tag®II:Strep-Tactin® system has provided powerful applications in the last 15 years for purification, detection and assay of recombinant proteins (reviewed in Schmidt & Skerra, Nature Protocols 2 (2007), 1528-1535) and even of cells (Knabel et al., Nature Medicine 8 (2002), 631-637).
The Strep-tag®II:Strep-Tactin® interaction is characterized by comparatively fast binding and dissociation kinetics and a medium binding affinity. Fast kinetics support higher flow rates during column chromatography where fast association kinetics ensures efficient binding and fast dissociation kinetics enables efficient competitive elution.
On the other hand, medium binding affinity and fast dissociation kinetics are limiting when at least one of the binding partners—Strep-tag®II fusion protein or Strep-Tactin®—is applied or present at low concentration. Examples for the first case are poor expression resulting in diluted extracts with respect to the Strep-tag®II fusion protein or using large buffer volumes for cell lysis after expression or secreting the Strep-tag®II fusion protein into the cell culture supernatant. In all examples, a large sample volume containing the target protein at low concentration needs to be applied to the affinity column often resulting in column breakthough, significant loss of Strep-tag®II fusion protein and reduced yield. The other variant of working under suboptimal conditions for this medium binding affinity interaction is diluting the Strep-Tactin® reaction partner as it is the case in batch purification which, as compared to column purification, equally may result in reduced yield for the Strep-tag®II fusion protein.
These limitations were reduced by developing the Di-tag affinity tag (similar or slightly different sequences are also known under the names Strep-tag®III, One-STrEP-tag or Twin-Step-tag®) consisting of a sequential arrangement of two (or more) Strep-tag®II moieties connected by a short linker. The linker and also the streptavidin binding moieties may be used in different variations. Examples of Di-tag sequences are the di-tag3 sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 103) or the di-tag2 sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 109) (Junttila et al., Proteomics 5 (2005), 1199-1203; U.S. Pat. No. 7,981,632). The biochemical reason for improved binding is the avidity effect, i.e. the combined synergistic binding of two streptavidin binding moieties to tetrameric Strep-Tactin®. This switches the comparatively fast off rate under non-competitive conditions to more steady binding while preserving efficient elution capability by adding a competitor that reverses the synergistic effect. In fact, the Di-tag features all beneficial application properties of Strep-tag®II, including efficient elution under competitive conditions, but additionally enables a more universal use in applications requiring more stable binding. One drawback is, however, that it has a considerably larger size (factor 3) than the short Strep-tag®II which makes adverse effects to the fused recombinant proteins more probable.
In addition to affinity purification, also assays may be quite demanding regarding binding affinity and dissociation kinetics, particularly when extensive washing is required. Thus, if a Strep-tag®II fusion protein to be analyzed is bound to a Strep-Tactin® coated solid phase, significant loss might occur during washing due to comparatively fast dissociation kinetics finally resulting in reduced sensitivity of the whole assay. Examples are ELISA or BiaCore™ or Quarz Crystal Microbalance (QCM) experiments where the recombinant Strep-tag®(II) fusion protein is immobilized on a microtitre plate or CM5 chip or sensor surface, respectively, each coated with Strep-Tactin®. The same is true, e.g., for applications where low amounts of a Strep-tag® (II) fusion protein, immobilized on a solid phase, are to be detected by Strep-Tactin® conjugated to a label in a sensitive manner. Examples are ELISA or Western blot or cell based assay experiments where the recombinant Strep-tag®(II) fusion protein is immobilized on or bound to a microtitre plate or membrane (nitrocellulose/PVDF) or a cell membrane, respectively. A cell membrane can also be considered as a solid phase as a bound Strep-tag®(II) protein can be, e.g., detected by labeled Strep-Tactin® via FACS. In fact, any detection method for a Strep-tag(II) fusion protein would be improved by a streptavidin mutein with increased binding affinity for the Strep-tag(II) or a Di-tag.
For these reasons a streptavidin mutein having a higher binding affinity for the short Strep-tag(II) than those muteins disclosed by U.S. Pat. No. 6,103,493 is still desirable. With such a streptavidin mutein, applications could be rendered possible using the short Strep-tag®II affinity tag which are currently only feasible by using the Di-tag. But also applications like purification, detection or assay for Di-tag fusion proteins would be enhanced by a streptavidin mutein with higher binding affinity for streptavidin binding peptides than the streptavidin muteins of U.S. Pat. No. 6,103,493. Examples for such applications are highly demanding situations in the purification applications described above or/and capture of diluted Di-tag fusion proteins in a batch format, e.g. capture of protein complexes with streptavidin mutein coated magnetic beads or/and in detection assays where highest sensitivity combined with extended washing is required. Such streptavidin muteins with enhanced affinities would also be desirable for most stable immobilization of fusion proteins carrying a streptavidin affinity tag such as the Strep-tag®II or the Di-tag, wherein these fusion proteins are to be characterized or assayed or detected on a solid phase, like, e.g., a chip for surface plasmon resonance (SPR), coated with said streptavidin muteins and wherein optionally said solid phase coated with said streptavidin muteins can be easily and under mild conditions be regenerated which means be deliberated again from the first Strep-tag®II or the Di-tag fusion protein to become ready for the binding of another Strep-tag®II or the Di-tag fusion protein.
It is therefore an object of the present invention to provide a streptavidin mutein having higher affinity than those muteins disclosed by U.S. Pat. No. 6,103,493 for streptavidin binding peptides such as the Strep-tag®II and/or Di-tag affinity peptide(s).